Project Summary The grey short-tailed opossum (Monodelphis domestica) is a small pouchless South American marsupial is a widely used model system for research into biomedicine and evolution. It is born prematurely, with well-developed forelimbs that permit it to climb to the mother's teat to complete its development. The hindlimbs, intervertebral disc, and a number of other organ systems are at a rudimentary stage of formation at birth. Because of the disparity in limb formation at birth, the opossum has long been used as a `natural' mutant for the study of limb formation. A number of evolutionary comparisons have been made between placental and marsupial mammals with this study system. In addition, the opossum has also been used in cancer research (particularly melanoma), studies of high cholesterol, spinal cord regeneration, wound healing, and birth defects (particularly thalidomide and retinoic acid teratogenesis), to name just a few. A number of resources have been developed for its husbandry, embryo collection, immunohistochemistry, in situ hybridization, and tissue culture. Its genome was also recently sequenced and organ-specific transcriptomes generated by RNA-Seq analysis. The opossum is also small-sized, fairly easy to raise in a laboratory setting, and relatively docile. Despite all these advantages, the main drawback to the marsupial model is a lack of transgenic or knockout animals. Transgenic mice have been available for decades, and become easier to make as technology improves. In addition, transgenic and other engineered mutants are becoming common for rats and other domestic mammals, particularly farm animals and non- human primates. Previous attempts to generate a transgenic opossum have had limited success. We propose to generate the first transgenic opossums based on a method that has been used to great success in other mammals, rodents especially. Spermatogonial stem cells (SSCs) from opossums will be harvested from the testes, cultured and expanded in vitro, transformed with a reporter-carrying construct, and transplanted into donor male opossums. If successful, this work will not only provide valuable comparisons of marsupial vs. placental mammal SSC biology, but will build the foundation for mutagenesis and transgenesis in an opossum model. Theoretically, it will be possible to utilize the increasingly popular CRISPR/Cas9 system in opossum SSCs and transplant them to donor males. For the many users of marsupial models, the availability of a transgenic marsupial model would be a critical addition to a growing toolkit. This proposal will be a proof-of-concept for the generation of opossums expressing exogenous DNA.